Brian J. Sweetman

Urinary prostaglandins. Identification and origin.

Human urine was analyzed by mass spectrometry for the presence of prostaglandins. Prostaglandin E2 and F2alpha were detected in urine from females by selected ion monitoring of the prostaglandin E2-methylester-methoxime bis-acetate and the prostaglandin F2alpha-methyl ester-Tris-trimethylsilylether derivative. Additional evidence for the presence of prostaglandin F2alpha was obtained by isolating from female urine an amount of this prostaglandin sufficient to yield a complete mass spectrum. The methods utilized permitted quantitative analysis. The origin of urinary prostaglandin was determined by stimulating renal prostaglandin synthesis by arachidonic acid or angiotensin infusion. Arachidonic acid, the precursor of prostaglandin E2, when infused into one renal artery of a dog led to a significant increase in the excretion rate of this prostaglandin. Similarly, infusion of angiotensin II amide led to a significantly increased ipsilateral excretion rate of prostaglandin E2 and F2a in spite of a simultaneous decrease in the creatinine clearance. In man, i.v. infusion of angiotensin also led to an increased urinary eliminiation of prostaglandin E. These results show that urinary prostaglandins may originate from the kidney, indicating that renally synthesized prostaglandins diffuse or are excreted into the tubule. Thus, urinary prostaglandins are a reflection of renal prostaglandin synthesis and have potential as a tool to delineate renal prostaglandin physiology and pathology.

Prostaglandins as Mediators of Hypercalcemia Associated with Certain Types of Cancer

We investigated the role of prostaglandins in the hypercalcemia associated with neoplasia. In patients with hypercalcemia and solid tumors the excretion of the major urinary metabolite of the E prostaglandins, 7 alpha-hydroxy-5, 11-diketotetranorprostane-1, 16-dioic acid (PGE-M), was significantly greater than normal, P LESS THAN 0.01 (median of 58.4 and 7.1 ng per milligram of creatinine respectively). Slightly elevated values were seen in normocalcemic patients with solid tumors (14.3 ng per milligram). The levels of the metabolite were normal in hypercalcemic patients with either hematologic neoplasia or primary hyperparathyroidism. Immunoreactive parathyroid hormone was undetectable in the plasma of all hypercalcemic patients with solid tumors. Inhibition of prostaglandin synthesis by aspirin or indomethacin reduced excretion of both the urinary metabolite and serum calcium in six hypercalcemic patients with solid tumors and elevated excretion of the metabolite. These findings support the concept that prostaglandins are mediators of the hypercalcemia caused by certain solid tumors.

Increased Production of Prostaglandin D2 in Patients with Systemic Mastocytosis

SYSTEMIC mastocytosis is a disorder of unknown origin, characterized by abnormal proliferation of tissue mast cells involving various organs and associated with a variety of clinical symptoms and s...

Increased arachidonate in lipids after administration to man: Effects on prostaglandin biosynthesis

Ethyl arachidonate was administered orally to 4 healthy male volunteers in a dose of 6 gm daily for a 2 to 3 wk period after a 1O‐day control period. The increased intake of this precursor of the dienoic prostaglandins resulted in significant increases in the relative and absolute amount of arachidonate in plasma triglycerides, phospholipids, and cholesteryl esters. Similar changes in lipid composition were noted in platelets. The excretion of 7 α‐hydroxy‐5, 11‐diketotetranorprostane‐1 ,16‐dioic acid, the major urinary metabolite of E prostaglandins in man, was increased by an average of 47% in 3 of the 4 volunteers. Platelet reactivity was assessed by determining the threshold concentration of adenosine diphosphate (ADP) necessary to induce secondary, irreversible aggregation of platelet‐rich plasma. This threshold concentration dropped significantly in all volunteers (10% to 60% of control values). It is concluded that the biosynthesis and function of prostaglandins can be augmented in man by oral administration of an esterified precursor fatty acid.

Relation between plasma concentration of indomethacin and its effect on prostaglandin synthesis and platelet aggregation in man.

The dose and plasma levels of indomethacin correlated with inhibition of prostaglandin synthesis as measured both by urinary excretion of the major metabolite of prostaglandin E2 (PGE‐M) and by the release of prostaglandin E drom thrombin‐stimulated platelets. Considerable intersubject variability was observed in the suppression of PGE‐M excretion. In some patients 37.5 mg indomethacin daily, usually considered subtherapeutic, caused suppression. Maximal suppression (>90%) occurred in some after a daily dose of 75 mg, whereas 150 mg was required to achieve this level of inhibition in others. Suppression of the excretion of PGE‐M by 60% occurred when the end of the dosage interval plasma levels of indomethacin were in the range 0.05 to 0.3 µg/ml, which implies that a somewhat higher average steady‐state concentration during the dosage interval was required to achieve this effect. A similar degree of inhibition of the release of PGE2 on thrombin‐stimulated platelets was associated with the same range of plasma levels. Upon discontinuation of the drug, the levels of indomethacin in plasma decreased exponentially; inhibition of the release of PGE2 from platelets by indomethacin declined linearly with time and in parallel with the logarithm of the diminishing plasma levels.

Biosynthesis of prostaglandin D2. 1. Formation of prostaglandin D2 by human platelets.

Formation of prostaglandin D2 (PGD2) during the aggregation of platelets was determined, employing a specific bioassay. PGD2 was synthesized in human platelet rich plasma (PRP) in response to thrombin, collagen and epinephrine. Indomethacin pretreatment abolished the biosynthesis of PGD2. When thrombin treated PRP was incubated for different periods of time and denatured in the presence of SnCl2 to prevent the formation of PGD2 from endoperoxides during the extraction procedure, PGD2 formation was noted within the first minute of incubation and reached a peak level after 4 minutes. PGD2 from thrombin stimulated PRP was conclusively identified by gas chromatography-mass spectrometry. The formation of PGD2 during platelet aggregation could represent a mechanism of feedback inhibition of aggregation.

Synthesis of prostaglandins by the rat renal papilla in vitro: Mechanism of stimulation by angiotensin II

1. The biosynthesis of prostaglandins in the rat renal papilla was studied in a whole-cell preparation in vitro. Prostaglandins recovered from the incubation medium were identified by gas chromatography-mass spectrometry as prostaglandin E2 and prostaglandin F2alpha. Quantitative estimates of prostaglandin output were obtained by bioassay and confirmed by selected ion monitoring. 2. Prostaglandin biosynthesis was enhanced by exogenous arachidonic acid and also by triglyceride lipase, indicating that arachidonic acid released from papillary triglycerides is readily available for prostaglandin biosynthesis. 3. Angiotensin II (10--100 ng/ml) stimulated the biosynthesis of both prostaglandin E2 and prostaglandin F2alpha, thus increasing prostaglandin levels in both the incubation medium and the tissues. 4. The mechanism whereby angiotensin II stimulates prostaglandin biosynthesis was investigated using the isotope dilution technique. In the presence of [14-C]-arachidonic acid, angiotensin II stimulated the output of more prostaglandin that had a significantly lower specific activity than the controls. Angiotensin II therefore increased the availability of endogenous, non-labelled substrate for prostaglandin biosynthesis. This conclusion was supported by experiments in which enough arachidonic acid was added to make the kinetics of prostaglandin synthesis zero order. Under such conditions angiotensin II failed to cause any further increase in prostaglandin synthesis. 5. It is concluded that angiotensin II controls prostaglandin biosynthesis in the renal papilla by regulating the availability of free precursor. Possible mechanisms for increased levels of free arachidonic acid could be the activation of a tissue acyl hydrolase or decreased utilization of fatty acids.

Quantification of the major urinary metabolite of the E prostaglandins by mass spectrometry: Evaluation of the method's application to clinical studies

Measurement of 7alpha-hydroxy-5,11-diketotetranoprostane-1,16-dioic acid, (PGE-M), the major urinary metabolite of prostaglandin E1 and E2 in man provides a useful indicator to monitor prostaglandin biosynthesis. For quantitative analysis of this prostaglandin metabolite and the stable-isotope dilution techniqe of selected ion monitoring (SIM) is employed using gas-liquid chromatography-mass spectrometry. The preparation of the bis(D3-methyloxime), bis-methyl ester of PGE-M containing a tritium tracer in position 2 which was used as internal standard for the SIM method is described. The synthesis of this internal standard includes the biosynthetic conversion of 11-hydroxy-9,15-diketoprostanoic acid to PGE-M by the rabbit. The intra-assay coefficient of variation of this SIM method ranged between 4.0 to 6.7 percent. The recovery of authentic, underivatized PGE-M added to urine was 93 +/- 3% (mean +/- SEM, n=17). The levels of PGE-M excreted in urine were higher (p less than 0.001) in males than in females (15.2 +/- 1.9 mug/24 hours (n=24) and 3.3 +/- 0.3 mug/24 hours (n=17), respectively. These levels were in close agreement with values published previously. No significant difference in excretion of PGE-M between the sexes was observed in the pre-pubertal age-grou (male: 2.9 +/- 0.8 mug/24 hours, n=5; female: 3.1 +/- 0.9 mug/24 hours, n=5) or in the age-group of 45-80 years (male: 9.3 +/- 1.1 mug/24 hours, n=21; female: 7.3 +/- 0.9 mug/24 hours, n=12). The amount of PGE-M excreted decreased significantly after administration of indomethacin or acetyl salicylic acid in therapeutic doses. The concomitant reduction of the urinary excretion of PGE-M (68 to 85% decrease) and prostaglandin E (73 to 100% decrease) after indomethacin treatment in each case (n=8) is evidence that a diminished urinary PGE-M output reflects a decrease in prostaglandin E biosynthesis.

Synthesis and metabolism of prostaglandins E2, F2α and D2 by the rat gastrointestinal tract. Stimulation by a hypertonic environment in vitro

Whole cell preparations of rat stomach corpus, jejunum, and colon were incubated and the released prostaglandin E2 (PGE2), PGF2alpha, PGD2, 15 keto-13,14 dihydro PGE2, and 15 keto-13, 14 dihydro PGF2alpha were measured by combined gas chromatography-mass spectrometry. All regions made PGD2 and possessed a high capacity for production 15 keto-13,14 dihydro derivatives of both PGE2 and PGF2alpha. Hypertonic sucrose solutions resulted in concentration-dependent increases in prostaglandin release, particularly of PGE2 and its metabolite. It is suggested that PGs may play a role in the local effects of luminal hyperosomolarity on digestive tract functions.

Effects of feeding ethyl-dihomo-γ-linolenate on prostaglandin biosynthesis and platelet aggregation in the rabbit

1. The ethyl ester of dihomo-gamma-linolenic acid (20:3omega6) (1 g/kg/day) was fed to rabbits for 25 days. Plasma lipids and platelet aggregation were analyzed on day 1, 11, 16, 21 and 26. 2. All plasma lipid classes were greatly enriched with 20:3omega6. Arachidonic acid levels were elevated to a smaller extent. The different platelet phospholipid fractions analyzed were also highly enriched with 20:3omega6, whereas the arachidonic acid content in platelet phospholipids was significantly lower than in control animals. 3. The excretion of 7 alpha-hydroxy-5,11-diketotetranorprostane-1,16-dioic acid, the major urinary metabolite of prostaglandin E1 and E2 was increased 4.6 fold by the treatment. 4. Platelet aggregation in response to ADP, collagen and arachidonic acid did not differ at any time betweeen 20:3omega6 treated rabbits and controls. 5. It is concluded that prostaglandin E biosynthesis can be increased by enriching the prostaglandin precursor pool. Platelet aggregation in vitro is not altered by feeding ethyl 20:3omega6.